Browse
Submit Data
Databases
API
Help

Dataset Information

34 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Linear-hairpin variable primer RT-qPCR for MicroRNA.

ABSTRACT: Here, we present a highly specific, sensitive and cost-effective system to quantify microRNA (miRNA) expression based on two-step RT-qPCR with EvaGreen detection chemistry, called linear-hairpin variable primer RT-qPCR. It takes advantage of the novel designed variable primer, which is initially designed to be linear, extending to form a hairpin structure and replacing the target miRNA for cyclic RT. Then the RT product is quantified by conventional EvaGreen based qPCR. The results show that this method has a dynamic range of 8 logs and the sensitivity is sufficient to directly detect down to 4 target miRNA molecules with a total analysis time of less than 2 hours. It is capable of discriminating between similar miRNAs, leading to an accurate representation of the mature miRNA content in a sample. The RT step can be multiplexed and the 8 miRNA profiles measured in 7 mouse tissues by this method show an excellent correlation with the commercial standard TaqMan RT-qPCR assays (r 2 = 0.9881).

SUBMITTER: Lan L

PROVIDER: S-EPMC6375362 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Linear-hairpin variable primer RT-qPCR for MicroRNA.

Lan Lin L Guo Qiuping Q Nie Hemin H Zhou Chang C Cai Qingyun Q Huang Jin J Meng Xiangxian X

Chemical science 20181204 7

Here, we present a highly specific, sensitive and cost-effective system to quantify microRNA (miRNA) expression based on two-step RT-qPCR with EvaGreen detection chemistry, called linear-hairpin variable primer RT-qPCR. It takes advantage of the novel designed variable primer, which is initially designed to be linear, extending to form a hairpin structure and replacing the target miRNA for cyclic RT. Then the RT product is quantified by conventional EvaGreen based qPCR. The results show that thi ...[more]

PMID: 30842860

Similar Datasets

RT-qPCR Testing of SARS-CoV-2: A Primer.

Project description:Testing for the presence of coronavirus is an essential diagnostic tool for monitoring and managing the current COVID-19 pandemic. The only reliable test in current use for testing acute infection targets the genome of SARS-CoV-2, and the most widely used method is quantitative fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR). Despite its ubiquity, there is a significant amount of uncertainty about how this test works, potential throughput and reliability. This has resulted in widespread misrepresentation of the problems faced using this test during the current COVID-19 epidemic. This primer provides simple, straightforward and impartial information about RT-qPCR.

| S-EPMC7215906 | biostudies-literature

Influence of RT-qPCR primer position on EGFR interference efficacy in lung cancer cells.

Project description:BACKGROUND:Real-time quantitative RT-PCR (RT-qPCR) is a "gold" standard for measuring steady state mRNA levels in RNA interference assays. The knockdown of the epidermal growth factor receptor (EGFR) gene with eight individual EGFR small interfering RNAs (siRNAs) was estimated by RT-qPCR using three different RT-qPCR primer sets. RESULTS:Our results indicate that accurate measurement of siRNA efficacy by RT-qPCR requires careful attention for the selection of the primers used to amplify the target EGFR mRNA. CONCLUSIONS:We conclude that when assessing siRNA efficacy with RT-qPCR, more than one primer set targeting different regions of the mRNA should be evaluated and at least one of these primer sets should amplify a region encompassing the siRNA recognition sequence.

| S-EPMC3047432 | biostudies-literature

Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets.

Project description:The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse transcription-PCR (RT-qPCR) assays are being used by clinical, research and public health laboratories. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer-probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT-qPCR analytical efficiency and sensitivity, we show that all primer-probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charité) confirmatory primer-probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes.

| S-EPMC9241364 | biostudies-literature

Human p53 signaling qPCR primer Array

Project description:H1299 cells were overexpressed miR-138 or silenced AGO2. The expression of 92 genes associated with p53 using the “Human p53 Signaling Pathway PCR Array” qPCR gene expression profiling. H1299 cells were transfected with NC mimics, AGO2 siRNA or miR-138 for 48h. Equal amount total RNA from each group was pooled prior to gene expression analysis.

2015-06-05 | E-GEOD-69562 | biostudies-arrayexpress

miPrimer: an empirical-based qPCR primer design method for small noncoding microRNA.

Project description:MicroRNAs (miRNAs) are 18-25 nucleotides (nt) of highly conserved, noncoding RNAs involved in gene regulation. Because of miRNAs' short length, the design of miRNA primers for PCR amplification remains a significant challenge. Adding to the challenge are miRNAs similar in sequence and miRNA family members that often only differ in sequences by 1 nt. Here, we describe a novel empirical-based method, miPrimer, which greatly reduces primer dimerization and increases primer specificity by factoring various intrinsic primer properties and employing four primer design strategies. The resulting primer pairs displayed an acceptable qPCR efficiency of between 90% and 110%. When tested on miRNA families, miPrimer-designed primers are capable of discriminating among members of miRNA families, as validated by qPCR assays using Quark Biosciences' platform. Of the 120 miRNA primer pairs tested, 95.6% and 93.3% were successful in amplifying specifically non-family and family miRNA members, respectively, after only one design trial. In summary, miPrimer provides a cost-effective and valuable tool for designing miRNA primers.

| S-EPMC5824350 | biostudies-literature

The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing.

Project description:BACKGROUND: The use of RNAi to analyse gene function in vitro is now widely applied in biological research. However, several difficulties are associated with its use in vivo, mainly relating to inefficient delivery and non-specific effects of short RNA duplexes in animal models. The latter can lead to false positive results when real-time RT-qPCR alone is used to measure target mRNA knockdown. FINDINGS: We observed that detection of an apparent siRNA-mediated knockdown in vivo was dependent on the primers used for real-time RT-qPCR measurement of the target mRNA. Two siRNAs specific for RRM1 with equivalent activity in vitro were administered to A549 xenografts via intratumoural injection. In each case, apparent knockdown of RRM1 mRNA was observed only when the primer pair used in RT-qPCR flanked the siRNA cleavage site. This false-positive result was found to result from co-purified siRNA interfering with both reverse transcription and qPCR. CONCLUSIONS: Our data suggest that using primers flanking the siRNA-mediated cleavage site in RT-qPCR-based measurements of mRNA knockdown in vivo can lead to false positive results. This is particularly relevant where high concentrations of siRNA are introduced, particularly via intratumoural injection, as the siRNA may be co-purified with the RNA and interfere with downstream enzymatic steps. Based on these results, using primers flanking the siRNA target site should be avoided when measuring knockdown of target mRNA by real-time RT-qPCR.

| S-EPMC3123217 | biostudies-literature

Human p53 signaling qPCR primer Array

Project description:H1299 cells were overexpressed miR-138 or silenced AGO2. The expression of 92 genes associated with p53 using the “Human p53 Signaling Pathway PCR Array”

2015-06-05 | GSE69562 | GEO

A novel and universal method for microRNA RT-qPCR data normalization.

Project description:Gene expression analysis of microRNA molecules is becoming increasingly important. In this study we assess the use of the mean expression value of all expressed microRNAs in a given sample as a normalization factor for microRNA real-time quantitative PCR data and compare its performance to the currently adopted approach. We demonstrate that the mean expression value outperforms the current normalization strategy in terms of better reduction of technical variation and more accurate appreciation of biological changes.

| S-EPMC2718498 | biostudies-literature

Analytical performance comparison of four SARS-CoV-2 RT-qPCR primer-probe sets for wastewater samples.

Project description:Current studies have confirmed the feasibility of SARS-CoV-2 RNA detection by RT-qPCR assays in wastewater samples as an effective surveillance tool of COVID-19 prevalence in a community. Analytical performance of various RT-qPCR assays has been compared against wastewater samples based on the positive ratio. However, there is no systematic comparison work has been conducted for both analytical sensitivity and quantitative reliability against wastewater, which are essential factors for WBE. In this study, the detection performance of four RT-qPCR primer-probe sets, including CCDC-N, CDC-N1, N-Sarbeco, and E-Sarbeco, was systematically evaluated with pure synthetized plasmids, spiked wastewater mocks and raw wastewater samples. In addition to confirm RT-qPCR results, Nanopore sequencing was employed to delineate at molecular level for the analytical sensitivity and reproducibility of those primer-probe sets. CCDC-N showed high sensitivity and the broadest linearity range for wastewater samples. It was thus recommended to be the most efficient tool in the quantitative analysis of SARS-CoV-2 in wastewater. CDC-N1 had the highest sensitivity for real wastewater and thus would be suitable for the screening of wastewater for the presence of SARS-CoV-2. When applying the primer-probe sets to wastewater samples collected from different Australian catchments, increased active clinical cases were observed with the augment of SARS-CoV-2 RNA quantified by RT-qPCR in wastewater in low prevalence communities.

| S-EPMC8464025 | biostudies-literature

qPCR primer design revisited.

Project description:Primers are arguably the single most critical components of any PCR assay, as their properties control the exquisite specificity and sensitivity that make this method uniquely powerful. Consequently, poor design combined with failure to optimise reaction conditions is likely to result in reduced technical precision and false positive or negative detection of amplification targets. Despite the framework provided by the MIQE guidelines and the accessibility of wide-ranging support from peer-reviewed publications, books and online sources as well as commercial companies, the design of many published assays continues to be less than optimal: primers often lack intended specificity, can form dimers, compete with template secondary structures at the primer binding sites or hybridise only within a narrow temperature range. We present an overview of the main steps in the primer design workflow, with data that illustrate some of the unexpected variability that often occurs when theory is translated into practice. We also strongly urge researchers to report as much information about their assays as possible in their publications.

| S-EPMC5702850 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data